Apoptosis is the genetically controlled death of unwanted or damaged cells during development and homeostasis. In recent years, a combination of genetic and biochemical approaches have been used to identify several families of molecules which act to facilitate or prevent apoptosis. Despite these advances in our knowledge of the basic machinery of apoptosis, we still know relatively little about how this process is regulated and executed during normal development. The long term goal of our research is to investigate developmental apoptosis using the powerful genetic and molecular techniques available in Drosophila. These studies will provide a foundation for understanding how apoptosis is misregulated in diseases such as neurodegeneration and cancer. The reaper (rpr), grim and hid genes act as central initiators of apoptosis in the Drosophila embryo. In the absence of all three genes embryonic apoptosis is blocked. Each of these genes induces caspase-dependent apoptosis when ectopically expressed. Current models for rpr and hid activity predict that apoptosis is initiated by physical interactions between Rpr, Grim and Hid with the apoptosis inhibitor DIAP1. Our preliminary data suggests-that this model is oversimplified. We propose to use a combination of genetic and biochemical strategies to test alternative models for Rpr and Hid interactions with DIAPI. The differential expression patterns of rpr, grim and hid suggest that they have unique functions in regulating developmental apoptosis. To examine the requirement for rpr, we have generated a mutant that removes the rpr g. Our preliminary characterization of this mutant has revealed a unique requirement for this gene in the programmed death of neuroblasts. Because the neuroblasts represent a defined population of cells that undergo apoptosis in a rpr-dependent manner, we can focus our investigations on the regulation of the apoptotic program in these cells. Not only are rpr, hid and grim essential for the initiation of apoptosis during development, they also are required for high levels of apoptosis in response to DNA damage in the embryo. Although Drosophila p53 (Dmp53) directly regulates rpr expression, our data indicate that deletion of rpr alone does not block Dmp53-induced apoptosis. In the rpr mutant background we will test other genes for their role in this death, and identify alternative targets of Dmp53. This work will improve our understanding of how p53 induces apoptosis.